Members of the transforming growth factor beta (TGF-β) superfamily of proteins are involved in embryonic development and adult tissue homeostasis. The TGF-β superfamily members share a common structure including a peptide signal sequence required for secretion of the protein and an amino-terminal fragment that is proteolytically cleaved about 105-140 amino acids from the carboxy-terminus of the large precursor protein to produce the mature protein. The mature protein is characterized by highly conserved cysteine residues, while the active form of the mature protein is a disulfide-linked homodimer of the proteolytically-cleaved proprotein (Gray, A., and Maston, A., Science, 247:1328, 1990).
Myostatin, also referred to as growth differentiation factor-8 (GDF-8) is a member of the TGF-β superfamily of proteins. Myostatin shares structural similarities with other TGF-β family members. It contains a hydrophobic amino-terminus that acts as a secretory signal and a conserved RSRR domain that is important for proteolytic processing. Cleavage of the protein gives rise to an amino-terminal latency associated peptide and a carboxy-terminal mature signaling peptide which forms the biologically active homodimer. Myostatin is expressed largely in developing and adult skeletal muscle and functions as a negative regulator of skeletal muscle. Systemic over-expression of myostatin in adult mice leads to muscle wasting (Zimmers, et al., Science, 296:1486-1488, 2002) while conversely, a myostatin knock-out mouse is characterized by hypertrophy and hyperplasia of the skeletal muscle resulting in two- to threefold greater muscle mass than their wild type littermates and a decrease in fat accumulation (McPherron, et al. Nature, 387:83-90, 1997). A human with a myostatin knock-out mutation was reported to be associated with gross muscle hypertrophy (Scheulke, et al., New Eng. J. Med. 350:2682, 2004).
There are presently limited treatments available for muscle wasting or for disorders or conditions which would benefit from an increase in muscle mass and/or muscle strength including, for example, muscular dystrophy, frailty, disuse atrophy and, cachexia, as well as disorders which are associated with muscle wasting, for example, renal disease, cardiac failure or disease, and liver disease. Due to its role as a negative regulator of skeletal muscle growth, myostatin is a desirable target for therapeutic or prophylactic intervention for such disorders or conditions or for monitoring progression of such disorders or conditions. Apart from its direct role in skeletal muscle regulation, myostatin may also be involved in other physiological processes including preadipocyte differentiation to adipocytes (Kim et al. BBRC, 281:902-906, 2001), and, indirectly with glucose homeostasis (McPherron, A and Lee S-J. JCI 109:595, 2002) and inhibition of bone formation (Hamrick, M. Mol. Cell. Evol. Biol. 272 388-91, 2003; Hamrick et al. Calcif Tissue Int. 71:63, 2002). Therefore, myostatin-specific antagonists, e.g., myostatin-specific antibodies, may also prove useful for treating, preventing or monitoring disorders or conditions such as those which benefit from increasing bone density (e.g., osteoporosis), Type II diabetes, metabolic syndrome, obesity and osteoarthritis.
Myostatin is highly conserved across species; the amino acid sequence of the mature form of myostatin in human, mouse, rat, chicken, turkey and cow are 100% identical (See FIGS. 2 and 3). There are naturally occurring myqstatin mutations in cattle, which have been linked to a double-muscled phenotype (McPherron, et al. PNAS, 94:12457-61, 1997). Since myostatin is highly conserved in sequence and in function across species, not only does an anti-myostatin antibody provide a promising means of increasing muscle mass, or treatment or prevention of such disorders or conditions listed above in humans, but also in other mammals including, e.g., domestic animals (e.g., canine and feline), sports animals (e.g., equine), food-source animals (e.g., bovine, porcine and ovine) and in avian species (e.g., chicken, turkey, duck and other game birds and poultry).
Growth differentiation factor-11, also referred to as GDF-11 or BMP-11, is the member of the TGF-β superfamily of proteins that is most homologous to myostatin. The amino acid sequence of the mature forms of human myostatin and GDF-11 are about 90% identical; however, GDF-11 is expressed in a wider range of tissues than is GDF-8 including dental pulp, brain, heart, kidney and lung as well as muscle and adipose tissue (Nakashima, et al. Mech. of Development 80:185, 1999). GDF-11 knock-out mice die within 24 hours of birth with multiple abnormalities. In particular the mice exhibit extra pairs of ribs, lack kidneys and show defects in the stomach, spleen and pancreas (McPherron et al., Nature Genetics 22:260, 1999; Esquela and Lee, Dev. Biol. 257:356, 2003; Harmon et al., Devpt. 131:6163, 2004). Human GDF-11 has recently been found to govern the temporal windows during which multipotent progenitors retain competence to produce distinct neural progeny (Kim, J. et al. Science 308:1927-1930, 2005).
There is a therapeutic need for an anti-myostatin antibody that preferentially binds myostatin over other TGF-β superfamily proteins, particularly GDF-11. Furthermore, there is a need for myostatin-specific antibodies which bind myostatin with a high affinity, particularly a higher affinity (i.e. a stronger affinity as shown for example by a lower KD value), than with which they bind GDF-11, and thereby allow the dosage level that patients receive to be minimized which may thereby result in less frequent dosing with such an antibody than with an antibody that binds myostatin with a lesser affinity (i.e., a higher KD). A high affinity antibody is also desirable in that it may allow for more flexibility in the route of administration of the antibody to a patient since it is less desirable for a drug to be administered intravenously than subcutaneously for example. There is also a need for myostatin-specific antibodies with a low or otherwise favorable IC50 value in a myostatin bioactivity assay in order to generate a therapeutic anti-myostatin antibody with a minimum effective therapeutic dose. It is also desirable to provide antibodies specific to myostatin where any immune response to the antibody evoked by a patient receiving the antibody is reduced to a minimum. The present invention satisfies these needs and provides related advantages.